In vitro diagnostic test for enterovirus infections

ABSTRACT

The present invention is related to an in vitro diagnostic assay of enteroviruses, based on the revealing of an immunologic reaction of antigen-antibody recognition type, using antigens or epitopes thereof that do not induce antiviral neutralising antibodies but induce “facilitating” antibodies which increase the viral infection.

The invention is related to an in vitro diagnostic assay ofenteroviruses based on the revealing of an immunologic reaction ofantigene-antibody recognition type.

Enteroviruses belong to the gender Enterovirus within the familyPicornaviridae. These viruses are of 25 to 30 nm of diameter,non-enveloped, with icosahedral symetry and a single-strand linear, notfragmented and positive RNA (ribonucleic acid).

The absence of the envelop endows them with a resistance tophysico-chemical agents and with a stability within the range of pH 3and 10. On the contrary, the enteroviruses are inactivated by heat at atemperature higher than 45° C. (and even 50° C. in the presence ofbivalent cations) and by major disinfectants and antiseptics (iodedprovidone, sodium hypochlorite, aldehydes).

There are 64 serotypes of enteroviruses: 3 polioviruses, 23coxsackieviruses A (CVA), 6 coxsackieviruses B (CVB), 28 echoviruses(EV) and 4 not classified enteroviruses.

More than 80% of the enterovirus genome of about 7500 nucleotides arecomposed of a unique reading frame flanked by two non-coding regions in5′ and 3′. The bulky protein encoded by this reading frame is cleaved in4 mature structural proteins VP1, VP2, VP3 and VP 4 (VP=viral protein)and in non-structural proteins, such as proteases and viralRNA-polymerase. A few animal enterovirus genomes were sequenced.Nevertheless, those which were sequenced, show a very high homology withhuman enteroviruses.

The enteroviruses are subject to a very high genetic variability due totranscription errors of the virus RNA polymerase, which are sources ofpunctual mutations, and to recombinations between different virusesgenomes. This variability contributes to the diversity of tissulartropisms and of the pathological spectra of enteroviruses.

The oral-faecal route is the primary way of transmission ofenteroviruses from person to person by contact of two individuals of thesame species or via water or contaminated food. Some serotypes aretransmitted by respiratory or cutaneous-mucous way. The enteroviruseswhich penetrate through the digestive tube first multiply in theintestine (hence the term enterovirus), before being spread throughoutthe body via the bloodstream toward the target organs (central nervoussystem, striated muscles, skin, . . . ). The non-apparent infections arethe most important part of enterovirosis. Acute infections andpersistent infections are distinguished among the symptomatic forms.

Human acute infections are highly polymorphous. Enteroviruses are themost frequent infectious agents responsible for central nervous systeminfections (lymphocytic meningitis, meningoencephalitis, paralysis ofpoliomyelitis type). They participate in numerous other respiratorypathological infections (rhinitis, bronchitis, bronchiolitis,pneumonia), heart infections (pericarditis, myocarditis), myositis,maculo-papulous or purpureous eruptions, febrile syndroms and, moreseldom, hepatitis, nephritis, orchitis or arthritis. Clinical symptomsare highly specific to enteroviruses and even to some serotypes;pleurodynia or Bornholm disease (CVB) corresponding to a kind ofhyperalgic influenza, vesicular eruptions of herpangina type orhand-foot-mouth syndrome (CVA, CVB, enterovirus 70), hemorrhagicconjunctivitis (CVA-24, enterovirus 70).

Animals, infected by enteroviruses are cattle, pork and poultry. Most ofthese enterovirus infections are not apparent and only porcine andaviary enteroviruses are responsible for economically importantdiseases. Some strains of porcine enterovirus are responsible forporcine polio-encephalomyelitis. The SVDV (swine vesicular diseasevirus) causes a porcine vesicular disease. Generally, the disease itselfis not severe and most of animals survive.

At least four chronic human pathologies account to be related toenterovirus: chronic meningo-encephalitis, post-poliomyelitic syndroma,heart affections and insulin-dependent diabetes. Indeed, strongarguments exist in favour of the coxsackievirus B implication ininsulin-dependent diabetes mellitus (IDDM) or type 1 diabetes. Severalauthors detected the presence of enteroviral RNA showing a high homologywith CVB in the peripheric blood of IDDM patients at the beginning ofthe clinical manifestations of the disease (Clements and al., 1995;Andreoletti and al., 1997; Nairn and al., 1999; Lonnrot and al., 2000).Recently, the Applicant has shown that high rates of interferon α (IFNα)in plasma are correlated in 50% of the cases with the presence ofenteroviral sequences, particularly CVB3 and CVB4, in circulating bloodof type 1 diabetic adults and children (Chehadeh and al., 2000). TheApplicant has also shown that CVB4, by interactions with circulating IgGantibodies or related to cells, can induce a high production of IFNα byperipheric blood mononuclear cells (PBMC) of IDDM patients (Hober andal., J. Gen. Virol., vol. 83, no. 9, September, 2002). Interferon αproduction is a marker of viral infection. This production is weaklyinduced by CVB4, except in the presence of the so called “facilitating”antibodies, which facilitate the viral infection. Therefore, CVB4 caninfect monocytes, mostly CD14+, by an antibody-dependent mechanism viainteractions between the virus, antiviral antibodies and specificreceptors on the cell surface (CAR, Fcγ RII, Fcγ RIII) resulting in IFNαproduction. This synthesis of IFNα induced by anti-CVB4 IgG reflects thepenetration of CVB4 into the monocytes but not the viral replication,and requires the presence of CVB4 RNA in the cells. If the IFNαproduction, induced by these anti-CVB4 IgG, is blocked, viral particlesproduced by PBMC can be detected (Chehadeh and al., J. Gen. Virol., vol.82, no. 8, August 2001). Moreover, the induction activity of plasma IFNαof IDDM patients preincubated with CVB4 before being brought to isolatedPBMCs of healthy subjects is high, compared with that in plasma fromhealthy subjects (Hober and al., J. Gen. Virol., vol. 83, no. 9,September 2002). IDDM patients have a higher prevalence of theseanti-CVB antibodies called “facilitating”, which enhance the CVB inducedIFNα synthesis in comparison with controls. Thus, the plasma of patientsinfected with enterovirus contains, besides of the presently knownneutralising antibodies which “immobilise” the virus and are mostlydirected to the epitopes of the structural surface protein VP1,“facilitating” antibodies favouring virus infection.

The diagnostic means used at present for diagnosis of enterovirosis aredirect means: cell culture, inoculation to new-born mouse, genomicamplification using primers in the non-coding 5′ region of the genome,and indirect means: sero-neutralisation and immunoenzymatic techniques.

Cell culture and seroneutralisation are not applicable to allcoxsackievirus A, the serotypes A1, A19 and A22 are not cultivable. Inthe practice, most of the CVA cannot be readily cultivated except theCVA9.

Inoculation to new-born mouse is a cumbersome and time-consumingtechnique, allowing to diagnose a coxsackievirus infection and todifferentiate between the CVA (flaccid paralysis) and the CVB (spasticparalysis).

Techniques of molecular biology, such as genomic amplification by PCR(Polymerase Chain Reaction), have permitted to detect low amounts ofenteroviruses by use of primers in highly conserved regions. However,they do not allow to detect all enteroviral infections, particularly ifthe infection is localised and the rate of virus replication is low,neither to differentiate one or another serotype. Immuno-enzymatictechniques also differentiate at the very most the groups and, beingbased on the detection of neutralising antibodies, they encountered theproblem of absence of a common antigen among enteroviruses.

Thus, there is no technique among these tests for detecting on a veryfine scale the serotype or variants (differentiation between wild andvaccinal strains), neither a method for quantification the viral load ininfected individuals.

In order to fill up this shortcoming, the Applicant has developed aspecific and quantitative in vitro diagnostic assay for enteroviruses.This assay is based on the existence of antigens inducing antibodies“facilitating” the virus infection and not neutralising antibodies.

Indeed, the Applicant identified the viral protein presenting theepitope or epitopes which are recognized by the “facilitating”antibodies in case of coxsackievirus B3 and B4 infection. This proteinis the structural internal protein VP4 and its demonstration was givenas follows

-   a) first, the viruses CVB3 and CVB4 E2 were cultivated and purified,-   b) then the proteins VP4 on one hand, and the H antigen (also termed    AEC for “artificial empty capsids”) on the other hand, were isolated    and purified from the dissociated viruses CVB3 and CVB4 E2,-   c) finally, it was shown that the VP4 protein inhibits the    enhancement of CVB/plasma couple induced IFNα production. Indeed,    when VP4 is preincubated with plasma before adding CVB, VP4 binds    the anti-VP4 antibodies and, therefore, less anti-VP4 antibodies are    left to bind the CVB and to facilitate the in vitro infection of    PBMCs and the enhancement of IFNα production by those. This proves    that the “facilitating” antibodies are anti-VP4 antibodies.

On the contrary, no cross reaction was observed between anti-VP4_(CVB3)and anti-VP4_(CVB4) antibodies. When VP4_(CVB4) was preincubated withplasma before addition of CVB3, no change in the CVB3 induced IFNα levelin the PBMCs cultures was observed. Neither the VP4 protein dissociatedfrom CVB3 affects the CVB4 induced IFNα level. Therefore epitopes ofthese two serotypes presented by the VP4 proteins are sufficientlydifferent, so that the “facilitating” antibodies will be specific to oneor another serotype.

This result was verified with the other CVB, CVB1 to CVB5 serotypes. Theanti-VP4_(CVB4E2) antibodies do not recognize the CVB virus of an otherserotype (Example 3).

The term “facilitating antibodies” denotes non-neutralising antibodieswhich facilitate the viral infection and are specific to the virusserotype, which antigen comes from.

The Applicant took advantage from this demonstration in order to developan in vitro diagnostic assay of enterovirus based on the revelation ofan immunologic reaction of antigen-antibody recognition type,characterised in that it uses antigens or epitopes thereof, which do notinduce antiviral neutralising antibodies but induce “facilitating”antibodies which increase the viral infection.

The problem of the present invention is related to a diagnostic assay ofenteroviruses, characterised in that either the antigens are fixed on asupport for the detection of the corresponding “facilitating”antibodies, or the “facilitating” antibodies are fixed on a support forthe detection of the corresponding antigens. The support is ofmulti-well microtitration plate or “chip” type, or any other support.The term “chip” denotes a miniaturised flat support of an inorganic ororganic solid material, such as glass, silicon or synthetic polymers, onwhich are bound polypeptides by covalent or non covalent binding.

When the assay of the invention aims the detection of “facilitating”antibodies by the corresponding antigens fixed on a support, thereaction is revealed by a labelled secondary anti-species antibody (forexample anti-human).

When the assay of the invention aims the detection of antigens by thecorresponding “facilitating” antibodies fixed on a support, the reactionis revealed by a labelled anti-viral antibody or by an anti-viralantibody (for example human), then a labelled secondary anti-speciesantibody (for example anti-human).

The marker of the secondary antibodies is preferably an enzyme, such asHRP (“horseradish peroxydase”) which shows a coloured or luminescentreaction with its substrate, a radioisotope, a fluorescent compound, achemiluminescent compound, a bioluminescent compound or a metal chelate.

It is preferable to use, as antigen inducing “facilitating” anti-viralantibodies, a full length viral internal protein or a fragment thereof.This protein can be either purified from the virus, can be a recombinantprotein presenting the same immunogenic properties, or can be obtainedby chemical synthesis and can present the same immunogenic properties.

In particular, it is preferable that the said viral internal protein bethe structural protein VP4.

In a particularly preferred manner, the antigen inducing the“facilitating” antiviral antibodies used in the diagnostic assay of thepresent invention is a peptide fragment of the VP4 protein taken amongthe peptides of the sequence SEQ ID NO1, SEQ ID NO2, SEQ ID NO3, SEQ IDNO4, SEQ ID NO5, preferably the peptide of the sequence SEQ ID NO2.

This assay particularly allows to diagnose a coxsackievirus type A or Binfection.

This assay comprises the following steps:

-   a—immobilisation of the “facilitating” antibodies or of the viral    protein inducing the “facilitating” antibodies or a fragment    thereof, on a support,-   b—immobilisation of control antibodies, of control proteins or a    fragment thereof, on a support,-   c—washing with a saline buffered solution supplemented or not with a    detergent in low concentration,-   d—saturation of the support surface not covered by a buffered    solution of irrelevant proteins,-   e—washing with a saline buffered solution supplemented or not with a    detergent in low concentration,-   f—adding of specimens to be studied at different dilutions in the    saturation buffer,-   g—washing with a saline buffered solution supplemented or not with a    detergent in low concentration,-   h—amplification of the response by use of labelled antibodies,-   i—washing with a saline buffered solution supplemented or not with a    detergent in low concentration,-   j—reading of the labelling intensity.

Particularly, the assay of the invention can be carried out by use of abox or a kit comprising:

-   -   antigens or “facilitating” antibodies of the invention,    -   reagents required for the constitution of the medium favourable        for performing the antigen-antibody reaction,    -   reagents allowing the detections of the formed complex.

According to the invention, the assay can be applied especially to thediagnosis of a CVB3 or CVB4 or any other CVB infection by detection anddosage of anti-VP4 antibodies by trapping them with the purified VP4protein fixed on a support. Indeed, the existence of a dose-responserelation between the amount of the VP4 protein pre-incubated with theplasma and the level of the IFNα production was revealed. Thus, theanti-VP4 antibodies of the plasma are trapped by the VP4 proteinpre-incubated with the plasma. The more is increased the amount of theVP4 protein in this assay, the more is diminished the amount of free“facilitating” anti-VP4 antibodies to recognize CVB, the more low is theIFNα production. Moreover, a dose-dependent correlation was foundbetween the amount of anti-VP4 antibodies pre-incubated with CVB3 orCVB4 before adding to PBMCs and the level of IFNα production. Noproduction of IFNα is detected in the presence of anti-VP4 antibodiesdepleted plasma or of irrelevant antibodies.

The assay object of the invention, allowing the dosage of the“facilitating” antibodies or of the viral protein carrying the epitopeor epitopes recognized by these antibodies, permits to measure theresponse of cells to viral infection, as for example the production ofIFNα.

The above described assay can be performed with other VP4 proteins ofother enteroviruses in order to carry out a screening of the infectioncaused by different viruses.

This assay can be used in case of a chronic disease related to anenteroviral infection aiming to establish the predictive value of theonset of the disease. More particularly, this assay can be used forpredicting the onset in pre-diabetes patients with a type 1 diabetesassociated with a CVB infection.

This assay can also be used for correlating the amount of detected“facilitating” antibodies and the stage of the disease. Moreparticularly, this assay allows to evaluate the stage of type 1 diabetesassociated with a CVB infection.

The diagnostic assay according to the invention can be used for thedetermination of the enterovirus serotype responsible for an acute humanor animal infection. Indeed, the “facilitating” antibodies and theantigen thereof are specific to a given enteroviral serotype and theassay of the invention can be multiplied by as many serotypes. To thiseffect, use can be made of as many supports as the serotypes, or to fixon a miniaturized support, such as a “chip”, for each serotype, the“facilitating” antibodies or the corresponding antigen.

Thus, the diagnostic assay of the invention can also be used for thedetermination of the distribution of enteroviral infections by serotypein a given human or animal population.

In a further embodiment of the invention, the diagnostic assay can beused for the determination of the viral target of an antiviral agent.Indeed, the antigen inducing “facilitating” antibodies can be determinedand dosed for each viral serotype by means of the diagnostic assay ofthe invention, by fixing the corresponding “facilitating” antibodies onone or more supports. Therefore, the in vitro infectivity of cells of avirus cultivable in the absence or presence of more or less importantconcentrations of antiviral agent can be measured. This can be performedalso for several serotypes and the target of the antiviral agent can bedetermined. In this embodiment, the diagnostic assay of the inventionallows to replace the long and tiresome measuring of the cell death invitro depending on the viral titer and on the concentration of theantiviral agent.

The following Examples illustrate the invention without limiting itsscope.

LEGEND TO THE FIGURES

FIG. 1: Effect of VP4 protein or H antigen on the CVB/plasma inducedIFNα production from 5 healthy subjects.

FIG. 1 a: Decrease of the dose-dependent. CVB3/plasma induced IFNαproduction in the presence of VP4_(CVB3) protein.

FIG. 1 b: Increase of the dose-dependent CVB3/plasma induced IFNαproduction in the presence of H antigen.

FIG. 1 c Decrease of the dose-dependent CVB4/plasma induced IFNαproduction in the presence of the VP4_(CVB4) protein.

FIG. 1 d: Increase of the dose-dependent CVB4/plasma induced IFNαproduction in the presence of H antigen.

FIG. 2: Comparison of the mean index values of anti-VP4 antibodies ofIDDM patients with healthy subjects.

FIG. 3: Mean IFN-alpha production by PBMCs after incubation of differentCVB serotypes with the plasma from healthy donors.

FIG. 4: Evaluation of the specificity of activating antibodies bymeasuring the IFNα production during the infection of PBMCS withdifferent serotypes of the virus in the presence of plasma obtained froma healthy donor (black), or in the presence of plasma pre-incubated withVP4 of CVB4B2 (hatched), using the DELFIA method.

FIG. 5: IFNα production during the infection of PBMCs with CVB4E2 orCVB3 in the presence of VP4 of CVB4B2 or of VP4 of CVB3.

FIG. 6: Plasmid pMAL-c2.

FIG. 7: IFNα production during the infection of PBMCs with CVB4E2previously incubated in the presence of plasma of healthy subjects andoptionally of recombinant VP4.

Legend to the FIG. 7

1. RPMI medium

2. Plasma

3. CVB4E2

4. VP4 1 μg/ml

5. Plasma+VP4

6. Plasma+CVB4E2

7. Plasma+VP4 0.01 μg/ml+CVB4E2

8. Plasma+VP4 0.1 μg/ml+CVB4E2

9. Plasma+VP4 1 μg/ml+CVB4E2

10. Plasma+VP4 10 μg/ml+CVB4E2

FIG. 8: Comparative ELISA Assay of VP4 and the peptides VP4-P1, VP4-P2,VP4-P3, VP4-F5, VP4-P6.

FIG. 9: Comparative ELISA Assay of VP4 and the peptide VP4-P2(patient=average of 40 serums of diabetic patients, control=average of40 serums of non diabetic patients).

FIG. 10: Comparative ELISA assay of recombinant VP4, VP4 and the peptideVP4-P2 (patient=average of 40 serums from diabetic patients,control=average of 40 serums from non diabetic patients).

FIG. 11 IFNα production during the infection of PBMCs with CVB4E2previously incubated in the presence of plasma from healthy subjects andoptionally of VP4.

Legend to the FIG. 11:

1: Medium

2: P2 (1 μg/ml)

3: Plasma 1/100th

4: CVB4E2

5: CVB4E2+Plasma 1/100th+Medium

6: CVB4E2+Plasma 1/100th+VP4(1 μg/ml)

7: CVB4E2+Plasma 1/100th+VP4-P2(1 mg/ml)

8: CVB4E2+Plasma 1/100th+VP4-P2(0.1 mg/ml)

9: CVB4E2+Plasma 1/100th+VP4-P2(0.01 mg/ml)

EXAMPLE 1 Revelation of the Viral Protein of CVB3 or CVB4 Inducing theAnti-CVB3 or Anti-CVB4 “Facilitating” Antibodies

a) Culture and Purification of the CVB3 and CVB4 Viruses

CVB3 (Americain Type Culture Collection, Manassas, USA) and diabetogenicCVB4 E2 (supplied by Ji-Won Yoon, Julia McFarlane Diabetes ResearchCenter, Calgary, Alberta. Canada) were propagated in Hep-2 cells(Biowhittaker, Verviers, Belgium) in Eagle's essential minimum medium(Gibco BRL, Eragny, France) supplemented with 10% of heat-inactivatedfoetal calf serum (Gibco BHL) and 1% of L-Glutamine (Eurobio, France).After incubation for 24 hours at 37° C., 5% CO₂, the cell suspension wasfrozen and thawed three times and centrifuged at 2000×g for 10 min.Virus obtained from the supernatant was pelleted by centrifugation at500 000×g for 3 hours at 4° C. in a Beckman TLA-100.4 rotor. The pelletwas resuspended in 3 ml of Tris-HCl 0.01 M pH 7.2, containing 0.5%(vol/vol) of Nonidet P40 incubated at 4° C. for 20 hours, homogenized,and centrifuged at 4000×g to remove the insoluble debris. Only 0.5 ml ofthe clarified virus suspension was layered on 0.5 ml of sucrose (30%,wt/v) and 3 ml CsCl (40%, wt/vol). After centrifugation at 348,000×g at4° C. in a Beckman TLA-100.4 rotor for 4 h, gradient fractions wererecovered and virus titers in gradient fractions were determined by the50% tissue culture infectious dose (TCID₅₀) assay on confluent cultureof Hep-2 cells. Fractions containing peak infectivity titers werepooled, dialysed and equilibrated with Tris-HCl 0.01M pH 7.2centrifugation at 4000×g on Macrosep™ membrane (Pall FiltronCorporation, Saint Germain en Laye, France) at molecular weight cutoffs(MWCO) of 300 K. Aliquots frozen at −80° C. were stored. Mockpreparations were obtained by the same protocol, except that the Hep-2cells were infected by the virus solvent alone.

b) Purification of VP4 Natural Protein and of H Antigen of CVB3 and CVB4Viruses

VP4 protein and H antigen were dissociated from the complete virusesCVB3 and CVB4 as described elsewhere for poliovirus (Maizel and al.,1967). Briefly, purified and concentrated CVB viral particles (˜1 mg)were incubated at 56° C. for 5 min in 0.5 ml sodium buffer (NaCl 0.1 M,sodium citrate 0.005 M, pH 7.0). This treatment results in thedissociation of the virus in viral RNA, VP4 and H antigen. Then VP4protein was separated from the mixture by centrifugation at 4000×g onMacrosep™ membrane at MWCO of 100 Kd. H antigen and RNA were retained bythe membrane, and VP4 passed through the membrane. RNA was degraded byadding 0.05 mg of bovine pancreatic ribonuclease A (Roche MolecularBiochemicals, Mannheim, Germany) and incubating at 37° C. for 10 min. Hantigen and VP4 were desalted and equilibrated with phosphate buffersaline (PBS), pH 7.2, by centrifugation at 4000×g on Macrosep™ membranesat MWCO of 100 Kd and 3 Kd respectively. H antigen and VP4 wereconcentrated using Savant Speed Vac Concentrator SVC100H (Global MedicalInstrumentation, Minnesota, USA). Mock dissociation was also made withsupernatant of non-infected Hep-2 cells purified as described above andyielded mock proteins for the following. The concentration of proteinswas calculated from the absorbance at 280 nm assuming an extinctioncoefficient of 1 mg/ml.

c) Inhibition of CVB/plasma Couple Induced IFNα Production by the VP4Protein

VP4, H antigen or mock proteins were preincubated for 1 h at 37° C. atdifferent concentrations with plasma from 5 healthy subjects diluted atoptimal dilution ( 1/10 or 1/100). CVB3 or CVB4 were then added for 1hour before incubating with PBMCs. As shown in FIG. 1, a dose-dependentdiminution of the CVB3/plasma induced IFNα production in the presence ofthe VP4_(CVB3) protein was observed, while in the presence of H antigen,no dose-dependent diminution of IFNα production was observed (FIGS. 1 aand 1 b). Similar pattern of results was obtained with VP_(CVB4) in thesystem CVB4/plasma (FIGS. 1 c and 1 d). On the contrary, the mockproteins isolated from non-infected Hep-2 cells have non effect on theIFNα production.

EXAMPLE 2 Diagnosis of a CVB3 or CVB4 Infection by Detection of Anti-VP4Antibodies—Relationship Between the Detection of Anti-VP4 Antibodies ofCVB3 and CVB4 and the Type 1 Diabetes

To detect anti-VP4 antibodies in donor plasma, microtitre plates of 96wells were incubated overnight at room temperature with the VP4 proteindissociated from CVB3 or CVB4 at 5 μg/ml in PBS pH 7.4. In the same way,microtiter plates were incubated with mock proteins. Then the wells werewashed three times with a washing solution (PBS pH 7.4, 0.05% Tween 20),saturated for 1 h at 37° C. with the saturation buffer (PBS, pH 7.4,2.5% skimmed milk, 0.5% Tween 20), and washed again 4 times. Then 0.1 mlof samples, diluted in saturation buffer at optimal dilution ( 1/50)were added to microwells. After incubating for 2 hours at roomtemperature, the wells were washed 4 times and 0.1 ml of IgA, G, Manti-human antibodies mixture labelled with HRP (“horseradishperoxydase”) diluted at 1/10000 were added and incubated for 1 h. After0.4 washing cycles, 0.1 ml of the substrate solution (0.4 mg/mlo-phenylen diamine, 0.012% H₂O₂ in phosphate citrate buffer 0.05 M, pH5.0) were added for 30 min. The reaction is stopped by addition of 25 μlof sulphuric acid 2 N. Absorbance measurement was carried out at 490 nmin a microplate reader Dynex MRX® (Thermo Life Science, Cergy-Pontoise,France). The immunologic assay cut-off value was determined by addingthe specimen absorbance on mock plates to that of the blank on VP4plates. Test specimens with an index (specimen/cut-off value ratio)greater than 1.0 were deemed positive for the presence of anti-VP4antibodies.

This assay was used for detection and dosage of anti-VP4 antibodies inplasma from healthy donors and IDDM patients. 14 out of 40 healthysubjects (35%) and 35 out of 40 IDDM patients (62.5%) were positive foranti-VP4_(CVB3) antibodies 6 out of 40 healthy subjects (15%) and 32 outof 40 IDDM patients (80%) were positive for anti-VP4_(CVB4) antibodies.The mean index value obtained was significantly higher than thatobtained in the group of healthy subjects (FIG. 2).

The assay developed in this example shows that the detection and thedosage of anti-VP4 antibodies can be used for the diagnosis of apathology associated to a coxsackievirus infection. Indeed, IDDMpatients present a more strong prevalence of anti-VP4 antibodies and ahigher amount of these antibodies than healthy subjects.

Some patients present a rate of anti-VP4 antibodies below the assay'sdetection threshold of the Example but, in fact, that can be explainedby the fact that their disease is related to other viruses than CVB3 orCVB4 such as CVB2.

EXAMPLE 3 Study of the Specificity of “Facilitating” Antibodies atSerotype Level

A) Materials and Methods

1—Production of IFNα by PBMCs in Culture

PBMCs separated from whole heparinated blood are distributed intomicrowells (plate of 96 wells), at the rate of 5 to 8.10⁵ cell in 100 μlof supplemented RPMI medium by well. The infection of PBMC is made witha final volume of 100 μl of preparation in each well. After incubationfor 48 h at 37° C. under a 5% CO₂ atmosphere and 90% humidity, thesupernatant is harvested and used immediately for IFNα dosage, orclarified by centrifugation for 10 minutes at 180×g and stored at −80°C. until dosage of the produced IFNα.

The concentration of the produced IFNα is determined with a sensitiveand specific technique, the DELFIA method (Dissociation EnhancedLanthanide FluoroImmmoAssay) (Rönnblom and al 1997).

2—Revelation of the Produced IFNα Immunofluorometric Method DELFIA

The dosage of IFNα is performed following the DELFIA principle(Dissociation Enhanced Lanthanide FluoroImmunoAssay), based on aimmunofluorescence in retarded phase method using antibodies bindingIFNα of which one is labelled with europium. The use of an activationsolution will release the europium conjugated with antibodies and emit afluorescence proportional to the amount of produced IFNα and measuredwith a fluorometer (fluorometer LKB Wallac 1230 ARCOSO®, Turku,Finlande).

Monoclonal anti-IFNα antibodies LT 273 (5.4 mg/ml) and LT 293 (4.8mg/ml) supplied by Dr Gunar Alm (Uppsala, Sweden) are coated to thebottom of the wells after an incubation for 12 hours at roomtemperature. These plates can be stored in a buffer at 4° C. or are usedimmediately. Standard samples of human IFNα are prepared with an“irrelevant” monoclonal mouse antibody IgG1 in order to establish thereference curve (10 measurements). The other samples to be analysed areadded to each well (100 μl) with the dilution buffer and the“irrelevant” antibodies and incubated for 2 hours with gentle stirringat room temperature. The wells are washed 3 times with the washingsolution. The antibody conjugated to europium (200 μl) is added to eachwell, left to incubate for 1 hour at room temperature with gentlestirring. The plate is washed 6 times with a washing solution. Theactivation solution (200 μl), added immediately after washing and leftfor 20 to 30 minutes for incubation in the well, causes a cleavage ofthe europium bound to the antibody fixed to IFNα, emitting afluorescence measured with the fluorescence reader (LKB Wallac 1230ARCUS®). The IFNα concentration will be calculated from the values ofthe measured fluorescence by means of the Graphpad program (San Diego,USA). The detection threshold of IFNα is of 0.5 IU/ml.

B) Results

1—Evaluation of IFNα Production by PBMC in the Presence of Plasmas andfor Each Serotype of Coxsackie B:

Donor plasmas (optimal dilution at 1/10th or 1/100th) are preincubatedfor 1 hour at 37° C. with different serotypes of the coxsackie virus B,CVB1 to CVB6 diluted at 1/10th. Then the peripheric blood mononuclearcells (PBMC) are infected. The revelation of the obtained IFNαproduction is carried out by DELFIA immunofluorometric method after 48hours infection of PBMCs. The same plasmas are used at identicaldilution for the study of the IFNα production by serotype. The amount ofPBMCs varies from 5.10⁸ to 7.10⁵/well. Plasmas from four differenthealthy donors were used.

Each CVB serotype causes an interferon alpha production in the presenceof plasma (see FIG. 3). No IFNα production is observed in the absence ofplasma in the experiences. Plasma dilution at 1/10th originates in aIFNα production in the average higher than the dilution at 1/100th(except for CVB6). On the contrary, the CVB1 virus, in the presence ofplasma, remains a weak inductor of IFNα.

2—Evaluation of Facilitating Antibodies Specificity for the Study ofIFNα Production During Infection of PBMCS by Different CVB Serotypes inthe Presence of VP4-CVB4E2:

Each CVB serotype was incubated one hour either with plasma alonediluted at 1/10th or with plasma diluted at 1/10th previously incubatedfor one hour with the VP4 recombinant protein (cf. Example 4) of CVB4E2diluted at 1/10th, either with MEM.

The results show, as previously, an IFNα production during the infectionof PBMCs in the presence of plasma. By preincubating of plasma with theVP4 protein of CVB4E2, this production of IFNα remains high and stablecomparing with previously obtained results, except in the presence ofCVB4E2 where the IFNα production has collapsed. The antibodiesfacilitating the induction of IFNα by CVB4B2 seem to be specificallydirected to the VP4 protein of CVB4E2. (see FIG. 4).

3—Evaluation of Facilitating Antibodies Specificity by Studying the IFNαProduction During CVB4E2 or CVB3 Infection of PBMCs in the Presence ofVP4 of CVB4E2 or VP4 of CVB3:

The plasma from a healthy donor (optimal dilution 1/10th or 1/100th) ispre-incubated for 1 hour at 37° C. with the recombinant VP4 protein ofCVB4E2 (optimal concentration at 1 μg/ml). The whole is incubated againfor one hour at 37° C. in the presence of the CVB4E2 virus (dilutions at1/10th or 1/100th; viral titer at 10¹³ TCID₅₀/ml) then applied to thePBMCs, (5.10⁵/well) to incubate for 48 hours. The IFNα production by thePBMCs is revealed by the DELFIA fluorometric method after the incubationfor 48 hours.

The same experience is performed with the same batch of plasma (optimaldilution), the natural protein VP4 of CVB3 (dilution at 1/10th) and theCVB3 virus (dilutions at 1/10th and 1/100th, viral titer at 10¹³TCID₅₀/ml). The determination of the IFNα amount produced by PBMCs isalso performed at 48 hours by DELFIA method.

Cross reactions are performed on the same principle in order to assessthe specificity of infection facilitating antibodies. The mixture ofplasma and VP4 of CVB4E2 preincubated for 1 hour is the incubated withCVB3 before being applied on the PBMCs for 48 hours.

In the same way, the plasma preparation preincubated for 1 hour and VP4of CVB3 are incubated for 1 hour with CVB4E2 before infecting the PBMCsfor 48 hours and determinating the amount of the produced IFNα.

The virus (CVB4E2 or CVB3) infected PBMCs in the presence of plasma aredeemed to be positive controls of this experimentation. The negativecontrols are obtained with one well of PBMC alone, one well of virusCVB4B2 alone and one well of CVB3 alone.

The virus CVB3 infection of PBMCs incubated for one hour with plasmafrom healthy donor leads to a IFNα production of 323 IU/ml (positivecontrol). When the same plasma from healthy donor was preincubated forone hour with the VP4 protein of CVB4E2 before being put in the presenceof the CVB3 virus for one hour, no difference is found in the IFNαproduction (308 IU/ml). On the contrary, when the same plasma isinitially preincubated for one hour with the VP4 protein specific toCVB3, before adding to CVB3, the IFNα production sinks to 6.4 IU/ml,which is 2% of its initial value (see FIG. 5).

The CVB4E2 virus infection of PBMCs incubated for one hour with plasmafrom a healthy donor leads to a IFNα production of 148 IU/ml (positivecontrol). When the same plasma from a healthy donor is preincubated forone hour with the VP4 protein of CVB3 before being put in the presenceof the virus CVB4E2, a high production of IFNα by the PBCMs was found(87 IU/ml). On the contrary, when the same plasma is initiallypreincubated for one hour with the VP4 protein of CVB4E2 before addingthe CVB4E2 virus, the IFNα production by PBMCs is low, 8.5 IU/ml.

The control viruses alone (CVB4E2 or CVB3), the proteins VP4 of CVB3 orof CVB4E2 alone, MEM or plasma alone do not lead to the IFNα productionby PBMCs.

EXAMPLE 4 Synthesis of Recombinant VP4 Protein of CVB4B2

A) Production of Recombinant VP4

1—Used Bacteria

Competent Escherichia coli bacteria JM 109 (Promega, Madison;United-States) were used.

2—Used Plasmid

The plasmid used for the transformation of competent bacteria is thepMAL-c2 (Valera-Calvino and al., 2000) supplied by Doctor Ruben ValeraCalvino, plasmid encoding a beta-lactamase, hence the selection ofbacteria which received the plasmid on an ampicilline-containing mediumand encoding the MBP protein, that was coupled with the VP4 protein,under the control of an IPTG inducible promoter (Promega, Madison,United-States). (see FIG. 6).

3—Used Media

α. SOC Medium

This medium comprises, per one liter, 20 g of Bacto™ tryptone(DIFCO-BECTON DICKINSON, United-States), 5 g of yeast extract (DIPCO,Detroit, United-States), 10 ml of NaCl 1M, 2.5 ml of KCl 1M, 10 ml ofMgCl₂ 1M/MgSO₄ 1M and 10 ml of glucose 2M, the pH is adjusted to 7.

β. LB Medium

This medium contains, per one liter, 10 g of tryptone (DIFCO-BBCTOMDICKINSON, United-States), 5 g of yeast extract (DIPCO, Detroit,United-States), 5 g of NaCl, the pH value is of 7.2. For dishes of LPmedium, this medium also comprises 15 g of ampicilline-containing agar(Invitrogen, Cergy Pontoise, France). The LB medium used for theproduction of recombinant proteins is enriched with an addition of 2 gof glucose per liter and comprises 100 μg of ampicilline per liter.

4—Buffers Used

α. Column Buffer

This solution comprises, per one liter: 20 ml of Tris-HCl (Q.Biogene,United-States) 1M pH 7.4, 11.7 g of NaCl, 2 ml of EDTA 0.5 M(Sigma-Aldrich, United-States). For elution of the MPB-VP4 protein, 3.6g of maltose (Sigma-Aldrich, United-States) (10 mM) are added to thecolumn buffer, then termed elution buffer.

β. Digestion Buffer

This solution comprises, per one liter: 3.2 g of Tris-HCl (Q.Biogene)(0.2 M), 5.84 g of NaCl (100 mM) and 0.22 g of CaCl₂, the pH is adjustedto 8.

5—Transformation of Bacteria

An aliquot of competent bacteria is thawed in ice, then 1 to 2 μl ofplasmid are added and incubated with the bacteria for 30 minutes in ice.Then the bacteria are subjected to a heat shock they are put at 42° C.for 30 s, before returning for 1 to 2 minutes into the ice. 250 μl ofSOC medium brought to room temperature are added, the tube is incubatedfor 1 hour at 37° C. with stirring. 20 to 100 μl of the bacteriasuspension are spread on a dish of ampicilline containing medium for theselection of transformed bacteria, the dish is incubated overnight at37° C.

Different volumes of the bacteria suspension are spread on differentdishes, in order to obtain at least one dish where the colonies areisolated. The dishes with colonies are stored at 4° C.

6—Production of the Recombinant MBP-VP4 Protein

10 ml of rich LB medium are inoculated with a colony of transformedbacteria in a tube Falcon® 15 ml, which is incubated overnight at 37° C.The next day, 1 liter of rich LB medium in a flask of 2.5 liters, isinoculated with these 10 ml, then incubated at 37° C. with stirringuntil its O.D. (optical density) attains a value in the range of 0.5 and0.6. Then the MBP-VP4 production is induced by addition of 3 ml of IPTG(isopropyl thiogalactoside) in the flask, which is incubated for atleast 3 hours at 37° C. with stirring.

7—Recovery and Obtention of the MBP-VP4 Protein

The culture of bacteria is centrifuged for 20 minutes at 4006×g at 4° C.The supernatant is then discarded and the bacteria pellet is taken up in50 ml of column buffer and frozen overnight at −20° C., before beingthawed in cold water and placed into ice the following morning. Bacteriaare lysed by 8 successive sonications for 15 seconds. The bacterialysate is clarified by centrifugation for 30 minutes at 900×g at 4° C.,the supernantant is recovered. Separately, 5 ml of amylose resin (NEWENGLAND BioLabs, United-States) are placed into the chromatographiccolumn, then washed with 40 ml of column buffer. The cell extractcontaining the fusion protein MBP-VP4 is injected into the column with aflow of 25.6 ml per hour. The resin is washed with 60 ml of columnbuffer. The MBP-VP4 protein is eluted with the elution buffer, thefractions are recovered at a rate of 1 ml per fraction. The fractionspresenting a maximum O.D. at 280 nm are pooled. The protein is desaltedagainst PBS by dialysis and concentrated on a filter Macrosep™ filtermembrane at 10 Kd (PALL, Life Sciences, United-States) by centrifugationat 5000×g, at 4° C., at least for 30 minutes.

The obtained MBP-VP4 protein is cleaved by the Factor Xa (NEW ENGLAND)BioLabs, United-States), which optimal concentration is of 2% of that ofthe fusion protein (determined by U.V. spectrometry with an absorbanceat 280 nm), in the elution buffer or digestion buffer.

The MBP protein, the not cleaved MBP-VP4 and the Factor Xa are removedby centrifugation on a Macrosep™ filter membrane at 10 Kd permeable onlyto the VP4 protein. The filtrate is recovered and concentrated bycentrifugation on a Macrosep™ filter membrane at 3 Kd (PALL, LifeSciences, United-States). The concentrated VP4 protein is stored at −80°C.

The CVIB4E2 induced IFNα production in cultures of PBMCs previouslyincubated in the presence of plasma from healthy subjects was measured.The IFNα production level is measured by DELFIA method in culturesupernatants recovered after 48 hours. Each culture condition requires2.10⁵ cell per well, and a m.o.i. of 1 for CVB4E2. Specimens of plasmawere diluted 1/10 tenfold in RPMI medium and incubated for 1 hour withCVB4E2 at 37° C. The recombinant VP4 protein is put into the presence ofplasma for one hour at 37° C. before incubation with CVB4E2. The resultsrepresent the means and the standard deviations of three independentexperiences performed with three different donors (see FIG. 7).

The recombinant VP4 protein, incubated with various amounts of plasma(10, 1, 0.1, 0.01 μg/ml), permits to note a dose-response effect of therecombinant VP4 protein on the effect exerted on the incubation withCVB4E2 of PBMCs by preincubation of plasma with the virus. Thus, withthe protein at a rate of 0.01 μg/mL a IFNα production of 189.5+/−21.6IU/mL, at a rate of 0.1 μg/ml 104.7+/−4.5 IU/mL, at a rate of 1 μg/ml44.1+/−24.3 IU/mL d'IFNα are obtained and at a rate of 10 μg/ml15.3+/−7.9 IU/mL are obtained. Thus, the more the dose of recombinantVP4 incubated with plasma is high, the more the values of IFNα are low(see FIG. 7).

EXAMPLE 5 Research of a Peptide Fragment of the VP4 Protein of CVB4E2Mimetising the Biological Activity of the VP4 Protein of CVB4E2 FullLength

A) Materials and Methods (ELISA Assay)

1—Assayed Serums and Plasmas:

40 plasmas from non diabetic patients and 40 plasmas or serums fromdiabetic patients were assayed.

2—VP4 Peptides Used in the ELISA Assays

The 5 VP4 peptides used were synthetized by the EPYTOPE Company (Nimes)on the basis of the sequence of the VP4 protein of the CVB4-E2 virus(listed in the NCBI data bank accession number of the sequence: Q86887).Their position on the sequence of the VP4 protein of the CVB4E2 virusand their sequences are presented in the Table 1 below. They areoverlapping and cover the totality of the sequence of VP4. TABLE 1 SEQID Name No Sequence Position VP4-P1 1 NH2-MGAQVSTQKTGAHETSLSAS-CONH2aa1-20 VP4-P2 2 NH2-GAHETSLSASGNSIIHYTNI-CONH2 aa11-30 VP4-P3 3NH2-GNSIIHYTNINYYKDAASNS-CONH2 aa21-40 VP4-P5 4NH2-NYYKDAASNSANRQDFTQDPSKFTEPVK aa31-60 DV-CONH2 VP4-P6 5NH2-SKFTEPVKDVMIKSLPALN-CONH2 aa51-693—Composition of the Used Buffers

PBS buffer: (for 1 liter): NaCl 8.0 g KCl 0.2 g KH2P04 0.3 gNa2HP04/2H20 1.44 g 

Adjust the pH to 7.4.

-   -   Washing solution

PBS pH=7.4 containing 0.05% Tween 20.

-   -   Saturation and dilution buffer:

Concentrated milk diluent/blocking solution (KPL) diluted at 1/20th inwater

-   -   Post-coating buffer:

Sodium phosphate buffer 50 mM pH=4.5 containing D-Sorbitol   6% NaCl0.9% BSA 0.1% CACl2/2 H20 0.1 mM EDTA  4 μM NaN3 0.005% 

Adjust the pH to 4.8

-   -   Secondary antibodies solution:

Anti-IgG.A.M antibodies (H+ L) human coupled to peroxidase (Bio-Rad)diluted to 1/10 000th in dilution buffer

-   -   Revealing buffer

Dissolve a capsule of phosphate citrate buffer (Sigma) in 100 ml ofdistilled water (Cf=0.05 M, pH 5.0). Take 25 ml of this buffer and placeinto a bottle of 50 ml wrapped in aluminium. Take a 10 mg ODP pastille(Sigma) and dissolve in these 25 ml (Cf=0.4 mg/ml). Mix using a vortex.Just before use add 10 μl of 30% H₂O₂.

4—Preparation of ELISA Plates

The VP4 protein or the 5 VP4 peptides, diluted at 5 μg/ml in PBS bufferpH=7.4, are coated in the wells of a microplate of 96 wells (FluoroMunc,MaxiSorp surface, NUNC, Danemark) at the rate of 100 μl/well. The platesare incubated for 1 hour at 37° C. under humid atmosphere, then left for24 h at room temperature. After 3 washings with PBS buffer, the wellsare saturated with 300 μl of saturation buffer for 1 hour at 37° C.under humid atmosphere, before washing 3 times with the washing buffer.Then 250 μl of post-coating buffer are added to each well and the platesare incubated for 24 h at room temperature, then stored at 4° C. untiluse.

5—Dosage ELISA:

The wells coated with the VP4 protein or the different peptides VP4 arewashed 3 times with washing buffer in order to remove the post-coatingsolution. Then 100 μl of the plasmas or serums diluted to 1/50th indilution buffer are added to different wells and the plates areincubated for 1 hour at 37° C. under humid atmosphere. Simultaneously, anegative control well containing 100 μl of dilution buffer is carriedout. Then the wells are washed 3 times with the washing solution and 100μl of secondary antibody solution are added to each well. Afterincubation for 1 h at 37° C. under humid atmosphere, the wells arewashed 5 times with the washing solution and 100 μl of substratesolution are added to each well. The plates are incubated for 30 minutesat room temperature and in the dark. The coloured reaction is stopped byaddition of 25 μl of H₂SO₄ 2M (1M) per well and the plates are read in aplate reader at a wavelength of 490 nm.

B) Results Obtained by ELISA from the Comparison of the VP4 Protein andthe Peptides VP4-P1, VP4-P2, VP4-P3, VP4-3P5, VP4-P6:

ODs obtained for each serum with the VP4 protein and the differentpeptides VP4 were divided by the OD obtained with the negative control.The following Tables show the mean +/− standard deviation of the ratesof OD obtained with the two series of assayed serums (Patient=diabeticpatients, Control=not diabetic). (see FIGS. 8 à 10) TABLE 2 Results ofELISA assay of VP4/peptides VP4 (see FIG. 8) VP4 VP4-P1 VP4-P2 VP4-P3VP4-P5 VP4-P6 Mean value 1.964 1.450 2.373 0.712 0.968 0.600 Standarddeviation 0.86466771 0.951 1.313 0.399 0.456 0.338

TABLE 3 Results of ELISA assay of VP4/peptide VP4-2 (see FIG. 9) Meanvalue (n = 40) Standard deviation VP4 Peptide P2 VP4 Peptide P2 Patient1.964 2.376 0.86466771 1.30832433 Control 0.711 0.691 0.348 0.23973485

TABLE 4 Results of ELISA assay of VP4/recombinant peptide VP4-2 (seeFIG. 10) Mean value (n = 40) Standard deviation Series VP4 VP4recPeptide P2 VP4 VP4rec Peptide P2 Patient 1.964 2.119 2.376 0.865 1.0091.308 Control 0.711 0.695 0.691 0.348 0.249 0.240

FIGS. 8 to 10 represent schematically the results of ELISA assays.

The peptides VP4-P1 (SEQ ID NO1) and VP4-P2 (SEQ ID NO2) show an ODcomparable to the VP4 protein in ELISA assay with 40 serums frompatients, (see FIG. 8).

Particularly, the peptide VP4-P2 (SEQ ID NO2) shows the same behaviouras VP4 in ELISA assay comparing the ODs of 40 serums from patients andof 40 serums from diabetic patients (see FIGS. 9 and 10). Thus thepeptide VP4-P2 (SEQ ID NO2), as VP4, recognizes a higher level ofantibodies with diabetics as with non-diabetics.

C) Results of Biological Test (IFNα Production by PBMCS) from theComparison of VP4 Protein and the Peptide VP4-P2

Material and methods for IFNα production by PBMCs in culture and forrevelation of IFNα produced by DELFIA method are the same as in Example3.

The peptide VP4-P2 (SEQ ID NO2) shows a diminution of the dose-dependentIFNα production during the infection of PBMCs in the presence of plasma.(see FIG. 11, tracks 7 to 9)

The peptide VP4-P2 mimetises therefore the VP4 protein in thisbiological assay.

These results, corroborated by ELISA assays (point B), suggest that thepeptide VP4-P2 (SEQ ID NO2) presents one or several epitopes recognizedby the “facilitating” anti-VP4 antibodies.

1. In vitro diagnostic assay of enteroviruses based on the revealing ofan immunologic reaction of antigen-antibody recognition type,characterized in that it uses antigens or epitopes thereof that do notinduce antiviral neutralizing antibodies but induce “facilitating”antibodies which enhance the viral infection.
 2. Diagnostic assayaccording to claim 1, characterised in that the immunologic reaction ofantigen-antibody recognition type is revealed either by a labelledanti-species antibody when the assay of the invention consists of thedetection of the “facilitating” antibodies by the corresponding antigensfixed on a support, or by a labelled anti-viral antibody or by ananti-viral antibody, then a labelled secondary anti-species antibody,when the assay of the invention consists of the detection of antigens bythe corresponding “facilitating” antibodies fixed on a support. 3.Diagnostic assay according to claim 2, characterized in that thelabelled antibody carries a label of an enzyme, a radioisotope, achemioluminescent compound, a bioluminescent compound or a metal chelatetype.
 4. Diagnostic assay according to claim 2, characterized in thatthe antigen inducing antiviral “facilitating” antibodies is a viralinternal protein.
 5. Diagnostic assay according to claim 4,characterized in that the viral internal protein is the full lengthprotein or a fragment thereof.
 6. Diagnostic assay according to claim 4,characterized in that the viral internal protein or a fragment thereofis obtained either by purification from the virus, or is a recombinantprotein presenting the same immunogenic properties, or is synthetizedchemically and presents the same immunogenic properties.
 7. Diagnosticassay according to claim 4, characterized in that the viral internalprotein is the structural protein VP4 or a fragment thereof. 8.Diagnostic assay according to claim 7, characterized in that thefragment of the structural protein VP4 is taken from the peptides ofsequences SEQ ID NO1, SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5. 9.Diagnostic assay according to claim 7, characterized in that thefragment of the structural protein VP4 is the peptide of sequence SEQ IDNO2.
 10. Diagnostic assay according to claim 1 or 9, characterized inthat the diagnosed enteroviruses are the coxsackievirus of A or B type.11. Diagnostic assay according to claim 1, characterized in that theperforming of the assay comprises a succession of the following steps:a—immobilisation of the “facilitating” antibodies or of the viralprotein inducing the “facilitating” antibodies, or a fragment thereof,on a support. b—immobilisation of control antibodies, of controlproteins, or a fragment thereof, on a support. c—washing with a salinebuffered solution supplemented or not with a detergent in lowconcentration d—saturation of the support surface not covered by abuffered solution of irrelevant proteins e—washing with a salinebuffered solution supplemented or not with a detergent in lowconcentration f—adding of specimens to be studied at different dilutionsin the saturation buffer g—washing with a saline buffered solutionsupplemented or not with a detergent in low concentrationh—amplification of the response by application of labelled antibodiesi—washing with a saline buffered solution supplemented or not with adetergent in low concentration j—reading of the labelling intensity. 12.Diagnostic assay according to claim 1, characterized in that theantigens or the corresponding “facilitating” antibodies are fixed on amulti-well plate type support for microtitration.
 13. Diagnostic assayaccording to claim 1, characterized in that the antigens or thecorresponding “facilitating” antibodies are fixed on a support of a chiptype.
 14. Box or kit for performing a diagnostic assay in vitro ofenteroviruses according claim 1, comprising: antigens or “facilitating”antibodies, reagents required for the constitution of the mediumfavourable for performing the antigen-antibody reaction, reagentsallowing the detection of the formed complex.
 15. Use of the diagnosticassay according to claim 1, for the prediction of the onset of a humanor animal chronic pathology related to an enterovirus infection.
 16. Useof the diagnostic assay according to claim 1 for the prediction of theonset of a type 1 diabetes in a pre-diabetic patient.
 17. Use of thediagnostic assay according to claim 1, for the definition of the stageof a chronic human or animal pathology related to an enterovirusinfection.
 18. Use of the diagnostic assay according to claim 1, for thedefinition of the stage of a chronic human or animal pathology relatedto CVB infection.
 19. Use of the diagnostic assay according to claim 1,for the determination of the enterovirus serotype responsible for anacute human or animal infection.
 20. Use of the diagnostic assayaccording to claim 1, for the determination of the distribution ofenteroviral infections by serotype in a given human or animalpopulation.
 21. Use of the diagnostic assay according to claim 1, forthe determination in vitro of the viral target of an antiviral agent.